ABSTRACT
BACKGROUND: ß-Hydroxy-ß-methylbutyrate (HMB) supplementation has been demonstrated to enhance muscle protein synthesis and attenuate loss of muscle mass by multiple pathways. The beneficial effects of HMB have been studied by using either the calcium salt, monohydrate, of HMB (CaHMB) or the free acid form (FAHMB). OBJECTIVE: The present study was designed to compare the pharmacokinetics and relative bioavailability of the 2 forms of HMB administered as a liquid suspension in male Sprague-Dawley rats. METHODS: CaHMB at 30, 100, and 300 mg/kg and equivalent doses of FAHMB at 24.2, 80.8, and 242 mg/kg were administered orally as a liquid suspension to male Sprague-Dawley rats. A single i.v. dose of 5 mg/kg CaHMB, corresponding to an equivalent dose of 4.04 mg/kg FAHMB, was also administered. Plasma concentrations of HMB were analyzed by liquid chromatography tandem mass spectrometry, and pharmacokinetic variables and relative bioavailability of the 2 forms of HMB were determined. RESULTS: After oral administration, the area under the plasma concentration time curve (AUC) from time 0 to time t (0-t) and from time 0 to infinity (0-∞) and the maximum (peak) plasma concentration (Cmax) for CaHMB were significantly greater than for FAHMB, whereas the time to reach Cmax did not differ from that of FAHMB. The relative bioavailability of CaHMB was 49%, 54%, and 27% greater than that of FAHMB for the 3 respective oral doses tested. After i.v. administration, the AUCs 0-t and 0-∞ of the calcium salt were significantly greater than those of FAHMB. The relative bioavailability of CaHMB was 80% greater than that of FAHMB. The higher relative bioavailability of CaHMB may be attributable to its low systemic clearance compared with FAHMB. CONCLUSIONS: This study demonstrates the enhanced relative bioavailability of CaHMB compared with FAHMB. Further studies are warranted to understand the physiologic mechanisms contributing to the differences in systemic clearance.
Subject(s)
Calcium/pharmacokinetics , Fatty Acids, Nonesterified/pharmacokinetics , Valerates/pharmacokinetics , Administration, Oral , Animals , Area Under Curve , Biological Availability , Calcium/administration & dosage , Calcium/blood , Dietary Supplements , Fatty Acids, Nonesterified/administration & dosage , Fatty Acids, Nonesterified/blood , Male , Microsomes/drug effects , Microsomes/metabolism , Protein Binding/physiology , Rats , Rats, Sprague-Dawley , Tandem Mass Spectrometry , Valerates/administration & dosage , Valerates/bloodABSTRACT
A simple, sensitive and specific high-performance liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of ß-hydroxy-ß-methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water-acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C(8) (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30-4600 ng/mL (r > 0.998) for HMB. The intra- and inter-day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench-top, autosampler freeze-thaw cycles and long-term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats.
